Tumor Location in the Head/Uncinate Process and Presence of Fibrosis Impair the Adequacy of Endoscopic Ultrasound-Guided Tissue Acquisition of Solid Pancreatic Tumors

Simple Summary Endoscopic ultrasound-guided tissue acquisition is the most accurate method to diagnose pancreatic tumors; nevertheless, this technique does not always bring adequate diagnostic accuracy. This study aimed to identify which factors can impair its adequacy. Pancreatic cytological and histological aspirates were retrospectively assessed according to two scores for grading the adequacy and the fibrosis of the specimens. The performance of the biopsies was lower when the tumor was located in the head/uncinate process of the pancreas, probably due to the higher fibrosis that we found in these sites. The specimens were less adequate also when <3 needle passes were performed and when the cell block was not done. We demonstrated the benefit to assess the presence of fibrosis in the specimens because it increased the risk of false negative results. Abstract Endoscopic ultrasound-guided tissue acquisition (EUS-TA) of solid pancreatic tumors shows optimal specificity despite fair sensitivity, with an overall suboptimal diagnostic yield. We aim to quantify the adequacy and accuracy of EUS-TA and assess predictive factors for success, focusing on the presence and degree of specimen fibrosis. All consecutive EUS-TA procedures were retrieved, and the specimens were graded for sample adequacy and fibrosis. The results were evaluated according to patients’ and tumor characteristics and the EUS-TA technique. In total, 407 patients (59% male, 70 [63–77] year old) were included; sample adequacy and diagnostic accuracy were 90.2% and 94.7%, respectively. Fibrosis was significantly more represented in tumors located in the head/uncinate process (p = 0.001). Tumor location in the head/uncinate (OR 0.37 [0.14–0.99]), number of needle passes ≥ 3 (OR 4.53 [2.22–9.28]), and the use of cell block (OR 8.82 [3.23–23.8]) were independently related to adequacy. Severe fibrosis was independently related to false negative results (OR 8.37 [2.33–30.0]). Pancreatic tumors located in the head/uncinate process showed higher fibrosis, resulting in EUS-TA with lower sample adequacy and diagnostic accuracy. We maintain that three or more needle passes and cell block should be done to increase the diagnostic yield.


Introduction
Endoscopic ultrasound-guided tissue acquisition (EUS-TA) is the safest and most accurate technique for obtaining tissue samples of pancreatic tumors [1,2]. However, the

Study Design and Patients
We performed a retrospective analysis of a prospectively collected database to retrieve all the patients with solid pancreatic tumors who underwent EUS-TA in the period 2007-2020 at the Hospital of Mantova, Italy. The study was conducted following the principles of the Declaration of Helsinki (revision of Edinburgh, 2000).
Inclusion criteria were EUS-TA for solid pancreatic tumor, age ≥ 18 years old, and signed informed consent for EUS and tissue acquisition. Exclusion criteria were cystic pancreatic lesions (regardless of the presence of solid components), previous attempts of tissue sampling, previous endoscopic retrograde cholangio-pancreatography with or without stent placement, and missing data about pathology or follow-up.
The following variables were recorded and analyzed: patients' gender and age, tumor size and location, needle type and size, number of needle passes, enrollment date, and preparation of the EUS-TA specimen.

EUS-TA
Antiplatelet and anticoagulant drugs were managed according to available guidelines. In detail, all agents except for aspirin were discontinued before EUS. All the procedures were performed by expert endosonographers (>1000 EUS procedures) under conscious sedation with meperidine and midazolam.

Material Preparation and Macroscopic Evaluation
As rapid on-site evaluation by a cytotechnician was not available, EUS-TA specimens were handled directly by the endosonographers. EUS-FNA material was slowly expelled by reinserting the stylet and a small drop of liquid was placed on each slide. The slides were smeared and macroscopically evaluated under oblique white light. The material was considered adequate when opaque thin granular or thread-like whitish material was visible and blood was either scarce or absent. Finally, the slides were fixed in a 95% alcohol solution. EUS-TA was terminated when at least five slides were deemed adequate according to the MOSE, regardless of the number of needle passes.
EUS-FNB material was expelled into a formalin vial by reinserting the stylet. The specimen was considered adequate if at least a 10 mm whitish core was floating in the liquid; otherwise, additional needle passes were repeated. The procedure was terminated when the specimen appeared sufficient according to the MOSE [26], regardless of the number of needle passes. Depending on the pathologist's preference, the cytological specimen was also used for cell block.

Pathologist Work-Up
Alcohol-fixed FNA-smeared slides were stained with Papanicolau (PAP) and May Grunwald-Giemsa (MGG) solutions. Formalin-fixed FNB tissue cores were processed as histopathologic samples.
Moreover, the alcohol used for the FNA slides and the formalin used for the FNB cores were visually inspected. If cloudiness or small fragments were seen, they were centrifuged in plastic test tubes for 4 min at 1500 rpm and the supernatant was decanted. A minimum of two thin smears were prepared from the sediment according to the PAP method. PAP and Hematoxylin and Eosin (HE) staining were done. The resulting pellets were fixed and overlaid with a 10% formalin-buffered solution. Pellets were placed in a cassette, embedded in paraffin wax, and finally trimmed in 3 µm thick sections for HE staining and immunocytochemistry (cell block technique).
Adequacy. In each pathology report, the proposed diagnosis together with the grade of specimen adequacy and tumor fibrosis were described. The adequacy was evaluated by assessing the presence of the neoplastic cellular component in the cyto-histological preparations, initially with low-power magnification (6.3× and 10×), then with highpower magnification (20× and 40×). Adequacy was graded according to the following classification: 0-inadequate (entirely or mainly represented by blood or contaminating gastric or duodenal mucosal flaps, with less than 100 neoplastic cells); 1-poor cellularity (just above the threshold of 100 pancreatic cells, visible only with high-power magnification); 2-moderate cellularity (cellular clusters visible in some low-power fields and many highpower fields); 3-rich cellularity (cellular clusters visible in many low-power fields and all high-power fields) [27] (Figure 1). 1-poor cellularity (just above the threshold of 100 pancreatic cells, visible only with high-power magnification); 2-moderate cellularity (cellular clusters visible in some low-power fields and many high-power fields); 3-rich cellularity (cellular clusters visible in many low-power fields and all high-power fields) [27] (Figure 1). Fibrosis. Since no standardized fibrosis score in EUS-TA pancreatic specimens was available, the grade of fibrosis was quantified according to a scoring system derived from breast fine needle biopsy [28].
Fibrosis was graded according to the following classification: grade 0 (total absence of fibrous tissue, desmoplastic stroma, or wispy collagen fibers in cytological smears or cell block; only the neoplastic cells are well represented); grade 1 (presence of 1-2 wispy collagen fibers at 40× magnification and/or presence of loose to moderate density fibrous tissue or desmoplastic stroma with an admixture of moderate cellular epithelial neoplastic component); grade 2 (presence of ≥3 wispy collagen fibers at 40× magnification and/or presence of dense fibrous tissue or well-evident desmoplastic stroma, with an admixture of less represented cellular epithelial neoplastic component) ( Figure 2). Fibrosis. Since no standardized fibrosis score in EUS-TA pancreatic specimens was available, the grade of fibrosis was quantified according to a scoring system derived from breast fine needle biopsy [28].
Fibrosis was graded according to the following classification: grade 0 (total absence of fibrous tissue, desmoplastic stroma, or wispy collagen fibers in cytological smears or cell block; only the neoplastic cells are well represented); grade 1 (presence of 1-2 wispy collagen fibers at 40× magnification and/or presence of loose to moderate density fibrous tissue or desmoplastic stroma with an admixture of moderate cellular epithelial neoplastic component); grade 2 (presence of ≥3 wispy collagen fibers at 40× magnification and/or presence of dense fibrous tissue or well-evident desmoplastic stroma, with an admixture of less represented cellular epithelial neoplastic component) ( Figure 2).
By definition, loose density stroma is characterized by a loose fibroblastic myxoid stroma and occasional short wispy collagen fibers. Moderate density stroma is composed of interrupted bands of keloid-like collagen (collagen with brightly eosinophilic hyalinization, similarly to keloid) without myxoid changes. High-density stroma presents mature collagen fibers, i.e., fine elongated collagen fibers densely packed into multilayers with intense staining lacking keloid-like collagen bands.
Final diagnosis-gold standard. The gold standard for the diagnosis was the pathological evaluation on the surgical specimens, when available. If surgery was not indicated, the data from EUS-TA results together with radiological and clinical follow-up were integrated.
In the case of non-diagnostic or inconclusive EUS-TA, patients were strictly followedup; the decision of repeating EUS-TA or performing other diagnostic and/or therapeutic interventions was based on the multidisciplinary evaluation. True negative EUS-TA was considered if the patients did not show any clinical, radiological, or histological signs of malignancy after at least one year of follow-up. By definition, loose density stroma is characterized by a loose fibroblastic myxoid stroma and occasional short wispy collagen fibers. Moderate density stroma is composed of interrupted bands of keloid-like collagen (collagen with brightly eosinophilic hyalinization, similarly to keloid) without myxoid changes. High-density stroma presents mature collagen fibers, i.e., fine elongated collagen fibers densely packed into multilayers with intense staining lacking keloid-like collagen bands.
Final diagnosis-gold standard. The gold standard for the diagnosis was the pathological evaluation on the surgical specimens, when available. If surgery was not indicated, the data from EUS-TA results together with radiological and clinical follow-up were integrated.
In the case of non-diagnostic or inconclusive EUS-TA, patients were strictly followed-up; the decision of repeating EUS-TA or performing other diagnostic and/or therapeutic interventions was based on the multidisciplinary evaluation. True negative EUS-TA was considered if the patients did not show any clinical, radiological, or histological signs of malignancy after at least one year of follow-up.

Statistical Analysis
Categorical variables were reported as number (no.) and percentage (%), while continuous variables as means ± standard deviation or median[interquartile range] when appropriate. Univariate and multivariate analyses were used to identify variables related to specimen adequacy, good adequacy (score 2 or 3), and risk factors for false negative EUS-TA results; odds ratio (OR) and 95% confidence interval (95% CI) were reported. Statistical analysis was performed using MedCalc ® Statistical Software version 19.5.2 (MedCalc Software Ltd., Ostend, Belgium; https://www.medcalc.org; accessed on 1 March 2020).

Statistical Analysis
Categorical variables were reported as number (no.) and percentage (%), while continuous variables as means ± standard deviation or median [interquartile range] when appropriate. Univariate and multivariate analyses were used to identify variables related to specimen adequacy, good adequacy (score 2 or 3), and risk factors for false negative EUS-TA results; odds ratio (OR) and 95% confidence interval (95% CI) were reported. Statistical analysis was performed using MedCalc ® Statistical Software version 19.5.2 (MedCalc Software Ltd., Ostend, Belgium; https://www.medcalc.org; accessed on 1 March 2020).

Study Population
Four hundred and seven patients were included. In all cases, the EUS-guided tissue acquisition was technically feasible. Baseline characteristics are shown in Table 1.
Pathology reports are described in Table 2.
In detail, a conclusive diagnosis was possible in 319 cases, while in 48 cases the pathological report was just negative for malignant cells, without any further specification. Finally, in 40 cases, the specimen was inadequate; of these, 20 were diagnosed with a pancreatic malignancy by further investigations and 20 were lost at follow up.    Sub-group analyses evaluating sample adequacy according to tumor location are shown in Tables 4 and 5.
Univariate and multivariate analyses of factors related to good sample adequacy (score 2 or 3) are shown in Table 6. Table 6. Variables related to good adequacy (score of 2 or 3) of EUS-guided tissue acquisition specimen.
Fibrosis was significantly higher for tumors located in the head/uncinate process, compared to the neck, body, and tail of the pancreas (Table 7). The lack of fibrosis was more frequently observed in the neck, body, and tail (71.1% vs. 54.8%), while low-to-moderate fibrosis (24.6% vs. 39.9%) and severe fibrosis (5.2% vs. 4.4%) were more frequently displayed in the head/uncinate process (p = 0.001). A lower fibrosis grade was also observed in EUS-TA with good adequacy, compared to samples with low adequacy (Table 7).

Diagnostic Performance
Overall  .8%]. In detail, 18 false negative cases were identified during follow-up and 1 false positive case was identified on the surgical specimen, after pancreatectomy. Table 8 shows variables related to false negative EUS-TA results. The subgroup of 48 patients that had a non-malignant biopsy showed peculiar characteristics: 35 (72.9%) of the lesions were located in the head/uncinate process; the mean adequacy rate was low (21 with adequacy score 1, 26 with score 2, and just 1 with score 3); a whatever degree of fibrosis (grade 1 or 2) was found in 15 (34.1%) out of 44 cases (in 4 cases the fibrosis was not evaluable).

Discussion
In this large single-center study, EUS-TA of solid pancreatic tumor emerged as a technically feasible and safe procedure, with overall adequacy of about 90%, including small lesions. For the first time, a four-level score measuring the degree of adequacy was applied to better analyze the variables influencing the performance of EUS-TA.
Our data showed that either a minimum of three needle passes or cell block were the most important technical factors increasing the adequacy of the aspirate, regardless of the type of the needle used. In our series, this combination provided a 98% adequacy [29].
We showed that the adequacy of EUS-TA was influenced by pancreatic tumor location. The procedures via a trans-duodenal route (towards the head/uncinate process) as compared to those via a trans-gastric route (towards the neck, body, and tail) had either an overall lower adequacy rate or a worse adequacy score. As the difference between the two puncture locations did not depend on the technical success of the procedure, we looked for other explanations.
Since other authors previously postulated that the stiff stroma of pancreatic tumors could decrease the performance of EUS-TA, we tried to quantify the fibrosis of the EUS-TA specimens to assess its possible relationship with the adequacy of the procedure. As it is hard to quantify precisely the fibrosis of a fine needle sample, we adapted a three-level grade from breast tumors to obtain an approximate quantification of the collagen fibers and the desmoplastic stroma. We then found two interesting facts. First, grades 1 and 2 of fibrosis were more frequent in the head/uncinate process (47.3% of cases) than in the neck, body, and tail (31.4% of cases), independently from the histotype of the lesion. Second, the presence of fibrosis, probably encasing the neoplastic cells in a dense, stiff matrix rich in fibroblasts, impaired not only the accuracy, but also the adequacy of EUS-TA samples.
These results had practical consequences as the head/uncinate process were either the most prevalent sites of tumors or the regions where the majority of poorly adequate and non-diagnostic biopsies occurred. Thus, we assume that to biopsy a focal lesion of the head/uncinate process in the absence of ROSE, it is extremely important to optimize our performance by performing at least three needle passes and the cell block. Additionally, we speculate that in the case of solid tumors with severe fibrosis, the use of ancillary techniques, such as contrast-enhanced harmonic guided tissue acquisition, could be used to increase the EUS-TA diagnostic yield [30].
Another meaningful information occurred when the specimen of a pancreatic focal lesion resulted in a non-malignant finding. These tumors were prevalently located in the head/uncinate process and showed a high fibrotic pattern and a low mean adequacy of EUS-TA. Moreover, these patients had a poor prognosis at follow up, showing all the same characteristics of pancreatic ductal adenocarcinoma. Thus, when a report comes back from the cytopathologist generically labeled as non-malignant, our suggestion is to consider that the degree of fibrosis is associated with a low accuracy of EUS-TA in these cases.
We acknowledge several limitations of our study. First, the two proposed scoring systems for adequacy and fibrosis have not been validated for EUS-TA. Therefore, our results should be verified by further investigations. Second, the long study period may have induced a temporal bias; however, we analyzed our results also according to the study period when EUS-TA was performed and found no significant difference in terms of adequacy and accuracy.

Conclusions
The adequacy of pancreatic EUS-TA was directly correlated with the performance of three or more needle passes and cell block but was negatively affected by the location of the lesion in the head/uncinate process. Presence and grading of tissue fibrosis were higher in lesions located in the head/uncinate process and seemed to be responsible for the negative impact on sample adequacy. We proposed two scores to quantify the degree of adequacy and grade of fibrosis of EUS-TA specimens; these results could stratify the risk of a false negative diagnosis. Further larger studies are recommended to confirm the low adequacy rate and the high prevalence of fibrosis in the head/uncinate process and to establish the applicability of our scores to EUS-TA.
Author Contributions: Conceptualization and methodology, T.T. and R.R.; software, T.T. and A.L.; formal analysis, A.L.; investigation, T.T. and R.R.; writing-original draft preparation, T.T., A.L., R.R. and P.F.; writing-review and editing, P.F. and A.F.; supervision, S.P. and N.P. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.

Institutional Review Board Statement:
The study was conducted in accordance with the Declaration of Helsinki; ethical review and approval were waived for this study due to its retrospective observational design.
Informed Consent Statement: Written informed consent for the EUS-TA procedures was obtained from all subjects involved in the study.

Conflicts of Interest:
The authors declare no conflict of interest.